During the development of a primary culture of chromaffin cells from adult rat adrenals, neurite outgrowth from chromaffin cells was sometimes observed. Subsequently we collected the media in which neurite outgrowth was observed in chromaffin cells and found that it stimulated neurite outgrowth activity. In this study, the culture conditions that lead to a generation of the neurite outgrowth promoting activity were established. We found that if dissociated adrenal medullary cells were first cultured in cytosine arabinoside-free media to allow non-chromaffin cells to proliferate and then in cytosine arabinoside containing media, the neurite outgrowth from chromaffin cells was observed. Furthermore, media collected one week after the addition of cytosine arabinoside were found to promote neurite outgrowth activity. The potency of the neurite outgrowth activity appeared to depend on the number of non-chromaffin cells in the culture, suggesting that non-chromaffin cells may be the source of the neurite outgrowth activity. This neurite outgrowth activity was found to be a protein, but is distinct from NGF, aFGF, bFGF, EGF, and IGF-I. We are currently preparing a large quantity of adrenal medullary cells for extraction of this neurite outgrowth promoting factors for further characterization.